This manuscript describes standard protocols for measuring tau hyperphosphorylation, measuring tau binding to microtubules, and localization of intracellular tau following drug treatments. These protocols can be used repetitively for screening drugs or other compounds that target tau hyperphosphorylation or microtubule binding.
The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning because it is involved in microtubule assembly and stabilization. Besides neurons, tau is expressed in human breast, prostate, gastric, colorectal, and pancreatic cancers where it shows nearly similar structure and exerts similar functions as the neuronal tau. The amount of tau and its phosphorylation can change its function as a stabilizer of microtubules, and lead to the development of paired helical filaments in different neurodegenerative disorders, such as Alzheimer's disease. Determining the phosphorylation state of tau and its microtubule-binding characteristics is important. In addition, examining the intracellular localization of tau is important in different diseases. This manuscript details standard protocols for measuring tau phosphorylation and tau binding to microtubules in colorectal cancer cells with or without curcumin and LiCl treatment. These treatments can be used to stop cancer cell proliferation and development. Intracellular localization of tau is examined by using immunohistochemistry and confocal microscopy while using low amounts of antibodies. These assays can be used repetitively for screening compounds that affect tau hyperphosphorylation or microtubule binding. Novel therapeutics used for different tauopathies or related anticancer agents can potentially be characterized using these protocols.
Tau was originally identified as a heat-stable microtubule-associated protein that was co-purified with tubulin1. Tau is exclusively expressed in higher eukaryotes2,3,4. The main function of tau is to control microtubule assembly1,5,6. It also contributes to polymerization of microtubules7, axonal transport8, changes in axonal diameter9, formation of neuroma polarity, and neurodegeneration10. Tau also acts as a protein scaffold to control some signaling pathways. Rat brain studies suggest that tau is neuron-specific and that it primarily localizes in axons11. Because tau is essential for microtubule polymerization and neuronal development, tau was hypothesized to play a major role in axonal development in the central nervous system; this hypothesis was later verified by in vitro and in vivo experiments. In addition to neurons, tau is expressed in different non-neuronal cells, including liver, kidney, and muscle cells12,13. Tau is expressed also in human breast, prostate, colorectal, gastric, and pancreatic cancer cell lines and tissues14,15,16,17,18,19. Tau is also found in inclusion-body myositis as twisted tubulofilaments in inclusion bodies20.
Tau may carry several post-translational modifications. Of all post-translational modifications, phosphorylation is the most common. Increased tau phosphorylation decreases its affinity for microtubules, finally destabilizing the cytoskeleton. Eighty-five phosphorylation sites have been described in tau protein isolated from human Alzheimer's disease brain tissues. Of these sites, 53% constitute serine, 41% threonine, and only 6% tyrosine residues21,22,23. Tau phosphorylation affects its localization, function, binding, solubility, and its susceptibility to other post-translational modifications. Also tau phosphorylation to more than the normal extent (or fully saturated with phosphate groups) is known as hyperphosphorylation that replicates structural and functional characteristics of Alzheimer's disease24. Tau maintains proper functioning of the axonal microtubules and ensures normal neuronal functioning under physiological conditions. However, hyperphosphorylated tau fails to maintain a well-organized microtubule binding, causing neuronal loss because of microtubule disassembly. Normal levels of tau phosphorylation are required for proper tau functioning, but tau fails to function normally if its characteristic phosphorylation level is altered and if it is hyperphosphorylated25. In Alzheimer's disease and some other age-related neurodegenerative disorders, tau becomes hyperphosphorylated and forms the paired helical filaments and neurofibrillary tangles26,27. Thus, methods for determining tau phosphorylation and microtubule binding are important.
Colorectal cancer, an ageing-associated cancer, is the third most frequently diagnosed cancer and the third prominent death-causing cancer for both men and women28. Colorectal cancer is one of the main death-causing cancers in the Western world29. Because both colorectal cancer and Alzheimer's disease are associated with ageing and both happen mainly in the developed countries where people enjoy similar dietary habits, the two diseases may somehow be correlated. In addition, tau-positive and tau-negative cancer cells respond differently to chemotherapeutic agents, e.g., paclitaxel16.
Curcumin is one of the main derivatives of Curcuma longa, the Indian spice turmeric30. For centuries, South Asian populations have consumed turmeric in their diets on a daily basis. Curcumin is used to treat different diseases, including colorectal cancer, Alzheimer's disease, diabetes, cystic fibrosis, inflammatory bowel disease, arthritis, hyperlipidemia, atherosclerosis, and ischemic heart disease31,32,33,34,35,36,37,38. Lithium can also kill colorectal cancer cells or prevent their proliferation39. Lithium can also be used for treating Alzheimer's disease40 as it decreases tau aggregation and prevents its hyperphosphorylation as observed in a transgenic mouse model41,42,43,44.
This manuscript aims to: 1) measure the total tau and phospho-tau expression levels in treated cells; 2) describe a phosphatase assay to measure overall tau phosphorylation; 3) examine microtubule-binding of tau; and 4) localize tau by confocal microscopy in colorectal cancer cell lines treated with curcumin or LiCl. Results reveal that cell treatment with curcumin, which is a supposedly good chemotherapeutic agent for colon cancer, and treatment with LiCl can reduce expression of both total tau and phosphorylated tau in colorectal cancer cell lines. These treatments can also cause nuclear translocation of tau. However, unexpectedly, curcumin fails to improve binding of tau to microtubules.
1. Preparation of Reagents
2. Cell Culture, Treatment with Curcumin or LiCl Treatment, and Examination of Protein Expression
3. Phosphatase Assay
4. Microtubule-binding Assay
Sample | Name | Microtubule needed | Main Protein needed | PEM-GTP-PTX | ||||
1 | HCT 116-Control (6.21 μg/μl) | 2 μl | 9.66 μl (60 μg) | 108.34 μl | ||||
2 | HCT 116-Curcumin 10 μM (4.81 μg/μl) | 2 μl | 16.63 μl (80 μg ) | 101.37 μl | ||||
3 | HCT 116-Curcumin 20 μM (3.28 μg/μl) | 2 μl | 30.49 μl (100 μg) | 87.51 μl | ||||
4 | HCT 116-LiCl 25 mM (5.43 μg/μl) | 2 μl | 18.42 μl (100 μg) | 99.58 μl | ||||
5 | E. coli tau | 2 μl | 1 μl tau-352 | 117 μl | ||||
6 | MT Only | 2 μl | X | 118 μl | ||||
120 μl each |
Table 1: Sample preparation for microtubule-binding assay.
5. Localization and Expression of Tau after Treatment of Cells with Curcumin
Expression of total tau and phospho-tau was examined after treating the cells with different concentrations of curcumin or LiCl (Figure 1). Treatment of cells with the three different concentrations of curcumin decreased tau expression levels; however, phospho-tau expression increased upon treatment with low concentration of curcumin but decreased upon treating cells with higher curcumin concentrations. Anti-phospho-tau (Ser396) was used for detection of phospho-tau. Levels of both total tau and phospho-tau decreased upon treatment of the cells with the three different concentrations of LiCl (Figure 1). Previous research had shown that tau expression levels vary in different cancer types and at different sites of the same cancer types. Previous data on colorectal cancer showed that tau was expressed in two cell lines (HCT 116 and SW480) of colorectal cancer that were also phosphorylated19. Phospho-tau levels in cells treated with 5 µM curcumin were higher than those in untreated cells. Phospho-tau levels were higher than total tau in cells treated with the same concentration of curcumin. Treatment of cells with 10 µM curcumin decreased phospho-tau levels compared with untreated cells but phospho-tau expression was still more than total tau expression levels. Treatment of cells with 30 µM curcumin lowered phospho-tau expression compared with phospho-tau in untreated cells and total tau levels in the same treated cells.
This manuscript established an easy protocol for assessing the phosphorylation status of tau by a phosphatase assay (Figure 2). Following curcumin treatment, overall tau phosphorylation did not change significantly and phosphorylation at specific amino acid residues was significant (Figure 1). Overall tau phosphorylation was stopped in cells treated with LiCl as compared with untreated cells. Phosphatase-treated samples electrophoresed faster than untreated samples, verifying that untreated samples were more hyperphosphorylated than phosphatase-treated samples. Curcumin-treated cells showed nearly the same results, indicating that treatment of colorectal cancer cell lines did not reduce tau phosphorylation as shown in Figure 1. Both phosphatase-treated and untreated samples electrophoresed at nearly the same range in samples of cells treated with LiCl, indicating that tau phosphorylation in these cells was reduced (Figure 2). Here, high concentrations (20 µM or 30 µM) of curcumin-treated samples were taken to compare overall phosphorylation status, as low concentration treatment showed higher site-specific phosphorylation revealed by specific phospho-tau (S396) antibody.
Moreover, in this lab, the microtubule binding assay of cell samples was established successfully using tau-352 as a positive control and only MT as a negative control (Figure 3). For both curcumin and LiCl treatment, microtubule-binding activity was inhibited, as demonstrated by the microtubule binding assay. In this experiment, curcumin treatment did not show microtubule binding capabilities but inhibited the binding similar to that in previous studies46,47, whereas it was effective to inhibit site-specific tau phosphorylation at higher concentration. In Figure 1, 10 µM curcumin treatment resulted in minimal expression of phospho-tau compared with control but higher expression than total tau. However, microtubule-binding capacity of colorectal cancer tau after curcumin treatment as well as low concentration of LiCl treatment was decreased in the untreated sample. Site-specific tau phosphorylation effects microtubule binding and self-aggregation48. Tau phosphorylation at proline-rich region impeded microtubule-binding properties whereas C-terminal region increased these properties, and both regions along with MT-binding region lessened its binding properties by about 70% and disordered the microtubules48. Some other factors as well as other site-specific phosphorylation of tau might be involved for this microtubule destabilization of colorectal cancer tau treated with curcumin or LiCl.
A straightforward protocol using small amounts of primary and secondary antibodies enabled localization of tau in colorectal cancer cell lines following curcumin treatment (Figure 4). Results showed that tau had translocated to the nucleus following curcumin treatment, a finding similar to earlier studies reporting that nuclear tau is a key player in neuronal DNA protection in neurodegenerative diseases such as Alzheimer's disease49,50.
Figure 1: Tau and phospho-tau expression in colorectal cancer cells following curcumin or LiCl treatment. Samples extracted from control cells or cells treated for 24 h with three different concentrations of (A) curcumin and (B) LiCl were resolved on 10% polyacrylamide gels by SDS-PAGE and probed with anti-tau or anti-phospho-tau antibody. Densitometry analyses of the Western blot results are presented on the right panel. Please click here to view a larger version of this figure.
Figure 2: Overall phosphorylation of tau in untreated or differentially treated cell samples detected by the phosphatase assay. Odd lanes show control cell extracts, whereas even lanes show phosphatase treatment of curcumin-treated or LiCl-treated samples. Phosphatase treatment made the tau electrophoretic mobility faster than untreated samples, which contained phosphorylated tau. Please click here to view a larger version of this figure.
Figure 3: Microtubule binding of tau in colorectal cancer cells detected by the microtubule-binding assay. Control HCT 116 cell samples, curcumin-treated, or LiCl-treated cell samples and E. coli tau were incubated with microtubules as detailed in protocol section 4. Equivalent amounts of supernatant (S) and pellet (P) fractions were immunoblotted with an anti-tau antibody. Negative control lanes containing only MT were run to confirm that MT did not contain any bound endogenous tau. Lower panel shows densitometry analyses of Western blots of individual samples enabling comparison between supernatant (unbound tau) and pellet (bound tau) fractions. Please click here to view a larger version of this figure.
Figure 4: Tau protein localization examined by confocal microscopy.
Tau localized peripherally to the nucleolus in colorectal cancer cells. Left panels show tau localization detected using the anti-tau monoclonal antibody whereas middle panels show nucleus staining by DAPI. Representative examples of treated cells showing tau translocation into the nucleus are also shown. Control cells showed tau mainly around and outside the nuclei, whereas tau in treated cells localized also inside the nuclei. Scale bar = 20 µm. Please click here to view a larger version of this figure.
This manuscript established different procedural conditions for detecting total tau and phosphorylated tau in colorectal cancer cells treated with curcumin and LiCl. To assess the overall phosphorylation status of tau in protein samples, a phosphatase assay was described. This assay can potentially be used to examine the phosphorylation status of any protein.
This assay is based on the principle that phosphorylated protein moves slower than its non-phosphorylated state. Alkaline phosphatase and alkaline phosphatase buffer are used in this protocol. After adding the assay components to the cell lysates, samples should be incubated for a specific optimal duration at a specific temperature. After incubation, samples should be boiled in the SDS sample buffer to stop the reaction. Previously, different experimental protocols may have been used to assess the binding of tau to microtubules. The microtubule-binding assay presented here is easy to perform while including both positive and negative controls to provide quality assurance using supernatant and pellet fractions. The MT-only negative control was used to verify that MT does not contain any bound endogenous tau. In addition, pure human tau-352, a microtubule-binding tau was used as the positive control. To separate the microtubule-binding fraction (pellet) from the non-binding fraction (supernatant) ultra-speed centrifugation was used; this procedure can be a limitation because such an ultracentrifuge is expensive and not widely available. In addition, because low quantities of samples are used, long, thin centrifuge tubes with adaptors are needed for use in the ultracentrifuge rotor. Though it is possible to re-use such tubes, single-use is advisable to avoid cross-contamination. Finally, an advantage of the tau-localization protocol is that only small quantities of primary and secondary antibodies are needed. After fixation, permeabilization, and blocking of the cells adherent to coverslips, a small drop of antibody was deposited on a small piece of PVDF, which was then placed inside the wells of a 6-well plate. The coverslips were kept such that the antibody could access the cells. By using this protocol, the amount of antibodies used can be minimized.
Some precautions should be kept in mind to ensure reliable and quantifiable results. Firstly, strict aseptic techniques should be used to avoid contamination in cell cultures and cell extracts. The complete RIPA lysis buffer should be prepared fresh. In the phosphatase assay, it is important to boil the samples for 3 min and start SDS-PAGE immediately after incubation of phosphatase-treated and untreated samples. Tau-352 should be divided into small aliquots and kept on ice after removal from the −20 °C freezer and deposited back into the freezer immediately after use. After removal from the −80 °C, MT stocks should be kept on ice if reused within 72 h; otherwise, keep them at room temperature. The PEM buffer should be prepared as a 10x concentrated solution because the 1x buffer needs to be freshly made when GTP and paclitaxel are added.
Tau functions are regulated by the extent of its phosphorylation. Tau hyperphosphorylation does not occur in normal adult brain tau similar to tau-352, which was used as positive control in the microtubule-binding assay. However, tau hyperphosphorylation occurs in neurodegenerative diseases. This protocol and subsequent results demonstrate that human colorectal cancer cell lines also carry phosphorylated tau similarly to that in Alzheimer's disease brains. Treatment with high-dose curcumin and especially LiCl can minimize tau phosphorylation. Thus, a good correlation between neurodegenerative diseases, such as Alzheimer's disease, and colorectal cancer may exist in regards to tau hyperphosphorylation, and curcumin or LiCl can be used to treat both colorectal cancer and Alzheimer's disease. Lastly, studying tau phosphorylation and tau microtubule binding is significantly relevant not only for neurodegenerative diseases but also for some chemotherapeutics because agents that modify tau microtubule binding are dynamically studied as cancer therapeutics51. The protocols presented here will potentially help the discovery and development of new therapeutic agents to treat different tauopathies and cancers.
The authors have nothing to disclose.
This research was performed as part of the project titled 'Development and industrialization of high value cosmetic raw materials from marine microalgae', funded by the Ministry of Oceans and Fisheries, Korea, and was supported by an intramural grant (2Z04930) from KIST Gangneung Institute of Natural Products.
HCT 116 cell | ATCC | CCL-247 | |
MEM (EBSS) | Hyclone | SH30024.01 | |
Fetal Bovine Serum (FBS) | ThermoFisher (Gibco) | 16000044 | Store at -20 °C |
penicillin-streptomycin | Hyclone | SV30010 | |
Trypsin-EDTA solution | WelGene | LS 015-01 | |
100 mm dish | Corning | 430161 | |
6 well plate | Corning | Coster 3516 | |
Anti-Tau 13 antibody | abcam | ab19030 | |
Dithiothreitol (DTT) | Roche | 10 708 984 001 | Storage Temperature 2–8 °C |
Microlitre Centrifuges | Hettich Zentrifugen | MIKRO 200 R | |
Paclitaxel | Sigma-Aldrich | T1912 | Storage Temperature 2–8 °C |
Curcumin | Sigma-Aldrich (Fluka) | 78246 | Storage Temperature 2–8 °C |
Microtubules (MT) | Cytoskeleton | MT001 | Store at 4 °C (desiccated) |
Mounting Medium with DAPI | Vector Laboratories | H-1200 | Store at 4 °C in the dark |
Sodium hydroxide | Sigma | 72068 | |
Magnesium Chloride | Sigma-Aldrich | M2670 | |
GTP | Sigma-Aldrich | G8877 | Store at -20 °C |
DPBS | WelGene | LB 001-02 | |
Sonic Dismembrator | Fisher Scientific | Model 500 | |
Ultracentrifuge | Beckman Coulter | Optima L-100 XP | |
PIPES | Sigma | P1851 | |
Bovine serum Albumin (BSA) | Sigma | A7906 | |
Molecular Imager | Bio-Rad | ChemiDoc XRS+ | Store at 4 °C |
Protein assay dye reagent | Bio-Rad | 500-0006 | |
α-tubulin (11H10) Rabbit mAb | Cell signalling | 2125 | |
GAPDH (14C10) Rabbit mAb | Cell signalling | 2118 | |
Anti-Tau (phospho S396) antibody | abcam | ab109390 | |
EGTA | Sigma | E3889 | Store at room temperature |
FastAP Thermosensitive Alkaline Phosphatase | Thermo Scientific | EF0651 | Store at -20 °C |
PMSF | Sigma | P7626 | Store at room temperature |
Phosphatase Inhibitor Cocktail | Cell Signalling | 5870 | Store at 4 °C |
Protease Inhibitor Cocktail | Cell Signalling | 5871 | Store at 4 °C |
RIPA Buffer | Sigma | R 0278 | Storage Temperature 2–8 °C |
Tau-352 human | Sigma | T 9950 | Store at -20 °C |
Triton X-100 | Sigma-Aldrich | X – 100 | Store at around 25 °C |
PVDF membrane | Bio-Rad | 162-0177 | |
Goat anti-mouse IgG Secondary Antibody | ThermoFisher | A-11005 | Store at 4 °C in the dark |
Confocal Microscopy | Leica Microsystem | Leica TCS SP5 | |
Sodium Dodecyl Sulfate (SDS) | Affymetrix | 75819 | |
Protein Assay | Bio-Rad | 500-0006 | Store at 4 °C |